Enzyme mediated synthesis of dipeptides is well known. Thus U.S. Pat. Nos. 4,165,311; 4,436,925 and 4,256,836 describe synthesis in aqueous medium to form insoluble addition compounds, e.g., the addition compound of one mol of phenylalanine methyl ester with one mol of N-protected aspartyl-phenylalanine methyl ester. U.S. Pat. No. 4,284,721 teaches that N-protected aspartic acid and phenylalanine lower alkyl esters can be enzymatically coupled in the presence of a water-immiscible solvent which can contain water-miscible co-solvents, but the amount of the water-miscible solvent must be limited to avoid inactivating or inhibiting the enzyme U.S. Pat. Nos. 4,116,768 and 4,119,493 contain similar teachings with regard to the use of water-miscible solvents as co-solvents in aqueous medium. Similarly Angew. Chem. Int. Ed. Engl. 24 (1985) Number 2, page 87, indicates that water-miscible solvents can be used as co-solvents in admixture with water but that the catalytic activity of protease enzymes decreases as the concentration of the co-solvent increases and that at 50% and above no synthesis occurs where chymotrypsin is used as the enzyme. As a possible exception, the use of a polyol (e.g., 1,4-butanediol) may in some cases stabilize the enzyme.
The use of aqueous or aqueous-organic media for enzymatic coupling to form N-formyl dipeptides (e.g., N-formyl aspartame) and polypeptides is also described in WO 8604924 and European Patent Publication 0149594.
A number of articles in various scientific journals also discuss the use of enzymes with a combination of water and water-miscible organic solvents and obtain yields which appear to vary depending upon the choice of solvent, amount of water, enzyme and substrate. Also, whether the enzyme is immobilized appears to be a factor. The use of a 50/50 acetonitrile/water solvent system is described by Nilsson and Mosbach in Biotech Bioeng. 26, 1146 (1984). This reference also describes the use of butanediol/water (90/10). The use of acetonitrile as a solvent is also discussed by J.B. Jones, and J.F. Beck, in Applications of Biochemical Systems in Organic Chemistry, Part 1 (J.B. Jones, C.J. Sih. and D. Perlman, eds.) p. 107 ff. New York; J. Wiley, 1976; and J.B. Jones and M.M. Mehes., Can. J. Chem. 57, 2245 (1979). The coupling of L-phenylalanine methyl ester (i.e., L-pheOMe) and N-protected N-carbobenzyloxy-aspartic acid (i.e., Z-asp) using a mixture of water-immiscible/water miscible solvents is described in Biotech. Lett. 7, 789 (1985). Konnecke et al. in Monatshrifts fur Chemie, 112, 469-481 (1981) at page 475 refer to the use of acetonitrile as a solvent. Other articles of interest are J. Biochem., 89, 385 (1981); J. Org. Chem., 51, 2728(1986); Coll Czechos. Chem. Comm., 49, 31 (1984); and Proc. Natl. Acad. Sci., 80, 3241 (1983).
While the literature indicates that enzymes in general and proteases in particular have been employed in both water-miscible and water-immiscible organic solvents, there appears to be a general conception that the water-miscible solvents are somewhat inferior. Thus it has been said that "most enzymes are inactive in hydrophilic, water-miscible organic solvents, which is easy to understand in terms of partitioning of the essential water from the enzyme into them." (A.M. Klibanov, Chemtech, page 354, June 1986).
This invention is based on the use of proteases in water-miscible organic solvents.